Treatments for chronic lung infections caused by nontuberculous mycobacteria (NTM), such as Mycobacterium avium-intracellulare complex (MAC) in immunocompromised people are both lengthy and complex, and recurrent infection with new strains of mycobacteria or caused by the original organism often occur. Evidence for multi- drug resistant (MDR) strains of another NTM, M. abscessus, have emerged in susceptible cystic fibrosis individuals. We hypothesize that host-directed immune responses induced by an immunotherapeutic vaccine regimen, may enable clearance of clinically important pulmonary infections, when given as an adjunct to drug treatment. We have developed a subunit vaccine (ID91), containing four different mycobacterial antigens (Rv3619, Rv2389, Rv3478, and Rv1886) which, when combined with a clinically tested, safe and synthetic toll-like receptor 4 (TLR4) agonist adjuvant (GLA-SE), shows promise against an aerosol infection with M. avium in a mouse model. In addition to the ID91 subunit vaccine, we have developed an innovative RNA- based vaccine platform that is purposefully designed to elicit a CD8+ T cell response (as opposed to our subunit vaccine that elicits a predominantly CD4+ T cell response) and will test this ID91-rvRNA vaccine for efficacy against M. avium. We believe that a prime/boost strategy, which engages several arms of immunity (including CD4+ and CD8+ T cells, and humoral responses) will provide more effective and long-lasting immunity against pulmonary mycobacteria such as M. avium. Importantly, our ID91 vaccine design uses bacterial antigens that share consensus sequences with BCG and different NTM strains (including M. avium), which could boost waning immune responses in immunocompromised individuals. Furthermore, we will leverage our extensive expertise obtained through the development of our first generation candidate vaccine ID93+GLA-SE, currently in Phase 2a clinical testing, to optimize and characterize the candidate ID91 vaccines, including the RNA vaccine. In Aim 1, we will test and characterize the efficacy of the protein and RNA ID91-based vaccines in a prime/boost strategy against M. avium in a mycobacterium-susceptible (and immunocompromised) mouse model (C3HeB/FeJ). Aim 2 will focus on the development of optimal prime/boost immunotherapeutic regimens, in combination with drug treatment using the C3HeB/FeJ mouse model. Efficacy and relapse rates for amikacin will be determined in M. avium infected mice following different lengths of drug treatment. Finally, we will test the optimal vaccine strategy (down selected in Aim 1) as an adjunct to the amikacin drug therapy regimen in the NTM C3HeB/FeJ mouse therapy model. Completion of the Aims in this proposal will lead to the development of experimental immunotherapeutic vaccines, as an adjunct to drug therapy, against pulmonary infection with M. avium, and will lead to the establishment of a novel NTM therapeutic mouse model for future testing of new drugs and experimental immunotherapy regimens.